The overall goal of this experiment is to detect enterohemorrhagic E.coli colonization in mice using a non-invasive in vivo imaging system. This method can help answer key questions in the enterohemorrhagic E.coli pathogenicity field such as colonization. The main advantage of this technique is that EHEC colonization can be detected in living animal without sacrificing animal.
To begin, streak a negative 80 degree Celsius stock of E.coli O157:H7 EDL933 bacteria harboring a luciferase plasmid onto an LB agar plate with 50 micrograms per milliliter of kanamycin. Grow the bacteria for 16 to 18 hours at 37 degree Celsius. Pick a single colony from the overnight plate and inoculate three milliliters of LB medium with 50 micrograms per milliliter of kanamycin.
Then, incubate the culture at 37 degree Celsius and 220 rpm for 16 to 18 hours. The following day, prepare a two milliliters of a one to 100 dilution of the overnight culture and inoculate it into 200 milliliters of LB with kanamycin. Incubate the culture at 37 degree Celsius and 200 rpm for 2.5 to three hours or until the optical density at 600 nanometers reaches between 0.9 and one.
Next, centrifuge the re-cultured bacteria at 8000 times gravity and four degree Celsius for 30 minutes. Discard the supernatant without disturbing the pellet and wash the bacteria with 100 milliliters of 0.9%sterile normal saline by gentle agitation. Following the wash, centrifuge the bacterial culture again and gently discard the supernatant.
Then, use 0.9%sterile normal saline to concentrate the pellet 100-fold. After treating mice with streptomycin water for 24 hours according to the text protocol, fill a syringe with 100 microliters of EHEC bacteria. Gently lift the animal and place it on the top of the cage with care.
Then, carefully grasp the mouse by the tail so that the animal grips onto the top of the cage and attempts to get away. Gently restrain the mouse by use the thumb and forefinger to grasp the loose skin of the neck and back of the animal to prevent the head from moving. Then, hold the mouse in a vertical position and ensure the head of the mouse is immobilized in this position.
Insert the gavage needle into the animal's mouth following the roof of the mouth and move down into the esophagus toward the stomach. When the inserted needle is half or two-thirds of the way in the mouse, inject the 100 microliters of EHEC bacteria which contains approximately 10 to the ninth CFU's of cells. After visualizing the bioluminescent cells according to the text protocol, open the Acquisition Control Panel of the software.
Select luminescent, photograph, and overlay. Set the exposure time to auto. Then set binning to medium.
Set the F/stop at one for luminescence and eight for photograph. F/stop controls the amount of light received by the CCD detector. Once the mice samples are ready for imaging, click acquire for image acquisition.
Open the image data that was acquired then open the tool palette panel. Select ROI tools such as the circle to define the bioluminescent area on the images. Next, using the pencil icon, click measure ROI's to measure the surface bioluminescence intensity.
The ROI measurement panel and the quantification values will appear. Then, on the left corner of the ROI measurements panel, use configure measurement to select the values/information needed. Otherwise, click Export to export this data table and save it as a csv file.
Finally use the values of the total flux p/s column as the bioluminescence intensity quantification in the csv file. As demonstrated in this image, a strong bioluminescence signal was seen in mice exposed to oral gavage with bioluminescence labeled EHEC inoculation. In this figure, mice inoculated with bioluminescence labeled wild-type EHEC EDL933 showed intense bioluminescent signals even at two days post-infection suggesting that EHEC colonized hosts within two days.
However, mice infected with a mutant E.coli with defects in lipopolysaccharides exhibit no bioluminescence suggesting that a reduced number or no bacteria colonized these mice. To localize the bioluminescence-labeled bacteria, the intestines of sacrificed animals were imaged ex vivo. The organs from mice infected with bioluminescence-labeled EHEC showed a significant bioluminescence signal in the cecum and colon indicating colonization in these organs.
In contrast, mice infected with an LPS mutant showed decreased bioluminescence in the intestinal tissue which is consistent with the in vivo image. After watching this video, you should have a good understanding of how to conduct the experiment using the IV system to monitor EHEC colonization in mice.
